A novel and inexpensive source of allophycocyanin for multicolor flow cytometry

Jou~al of lmmunolo~cal Methods, 121 (1989)9-18

9

Elsevier

JIM 05212

A novel and inexpensive source of aUophycocyanin for multicolor flow cytometry T h o m a s M. Jung a n d Morris O. Dailey Departments of Pathology and Microbio!o~,y, Universityof lowa, Collegeof Medicine, Iowa City, IA, U-S.A.

(Received3 October1988, revisedreceived13 February1989, accepted14 February1989)

Allophycocyanin (APC) belongs to a family of phycobiliproteins that are well suited as fluorescent reagents for flow cytometric analysis, since they halve a broad excitation spectrum, a large Stoke's shift and they fluoresce with a high quantum yield. The widespread use of APC has been 1/mired by the availability of raw material and high cost of the purified phycobiliprotein. We have assessed the suitability ~f dry, powdered Spirulina platensis, available at health food stores, as an inexpensive source of APC. APC was extracted from Spirulina platensis by overnight treatment with lysozyme, followed by ammonium sulfate precipitation. APC was then separated from phycocyanin (the only other, major phycobiliprotein in Spirulina) by elution of bound material from an hydroxylapatite column using an increasing continuous phosphate gradient. APC isolated in this manner retained its norms" u,/meric structure. The absorbance and fluorescence excitation and emission spectra of the purified phycobiliproteius were identical to those previously shown for C-PC and APC. APC can be stored concentrated at 4°C, frozen at - 7 0 ° C , or as a saturated ammonium sulfate precipitate, with no subunit dissociation or change in spectral properties. Moreover, APC has been conjugated to monoclonal and polyclonal antibodies for use in multicolor FACS analysis, with the conjugated antibody activity rema/ning stable for at least 2 years. Thus, this procedure is a simple, cost-effective method for preparing reagents for multicolor immunofhiore~ence and flow cytometry. Key words: Allophycocyanin;Phycobiliprotein;Flow cytometry;Immunofluorescence;Receptor

Introduction Immunofluorescence analysis of multiple cell populations has been aided by the use of precisely Correspondence to: T.M. Jung, 365 MRC, Department of Pathology,Universityof Iowa, Iowa City, IA 52242, U.S.A. Abbreoiations: APC, allophycocyanin;BSA, bovine serum albumin; FACS,fluoreseence-activ~tedcell shorter; FITC, fluo ~ isothiocyanate;2-1T, 2-iminothiolane;PbP, phycobiliprotein; PBS, phosphate-bufferedsaline; PC, phycocyanin; PCB, phycocyanobilin;PE, phycoerythrin;PEB, phycoerythrob'din; PUB, phycourobilin; SMPB, succinimidyl d.(pmaleimidoplienyl)butyrate.

defined monoclonal antibodies to cell surface antigens. Simultaneous staining of cells with three and four different monoclonal antibodies coupled to separate fluorochromes identifies multiple cell subsets based upon their distinct patterns of antigen expression. Phycobiliproteins are fluorescent accessory photosynthetic pigments which have been used to label antibodies for fluorescenceactivated cell sorter (FACS) analysis, These proteins serve to transfer energy from blue fight to the chlorophyll photosynthetic system and are quite abundant in cyanobacteria and red algae (Z~!inskas et al., 1980; Csatorday et al., 1988). The

0022-1759/89/$03.50 © 1989 ElsevierSciencePublishersB.V. (BiomedicalDivision)

phyce.biliproteins are well suited for use in multicolor fluorescence analysis because they have broad excitation spectra, a large Stoke's shift and they fluoresce with a high quantum yield (Oi et al., 1982; Glazer and Stryer, 198,*). In addition, since these fluorochromes emit at long wave-lengths, they are often better suited for anatysis of surface antigens on autofluorescent cells, since most autofluorescence emission occurs at shorter wavelengths (Alberti et al., 1987). There are three families of phycobiliproteins, the phycoerythrins (PE), the phycocyanins (PC) and the allophycocyanins (APC). These fluorochromes have a protein core composed of two homologous polypeptide chains (a,/3), which are present in equimolar amounts (Brown and "Iroxler, 1977; Offner et al., 1983; lsono and Katch, 1987). The differences in spectral properties between phycobiliproteins result largely from the different number and types of bilin prosthetic groups attached to the core. The phycoerythrins are usually isolated as hexamers ((a, fl)6) and have the 'red' chromophore phycoerythrobilin (PEB) as the major prosthetic group. R-PE is isolated from red algae, whereas B-PE is from a bacterial source. Phycourobilin (PUB) is present in B- and R-PE and is responsible for its 495-500 nm absorption peak. The phycocyar&ls (PC from a red algae source and C-PC from cyanobaeteria) exist either as a trimer ((a, ~8)3) or hexamer ((a. fl)6) and ~ontain either a mixture of the 'blue' chromophore phycocyanobilin (PCB) and PEB or just PCB as prosthetic groups, depending on the species of origin. Allophycocyanin is a (a, ~8)3 trimer and has PCB as prosthetic groups (for a review see Glazer, 1982). APC and PC have been shown to be particularly useful in flow cytome'iry and cell sorting, using either tunable dye lasers or the less expensive, higher wavelength helium-neon lasers (Shapiro et al., 1983; Parks et al., 1984; Loken et al., 1987). Because the higher emission wavelength of APC is well separated from that of Texas Red, it can also be used for four-color FACS analysis. Although the utility of APC has clearly been demonstrated, its widespread use has been somewhat limited by its high cost and the poor availability of raw material from which it can be isolated.

We report here on a unique and easily accessible source of the cyanobacteria, Spirulina platensis, from which one can isolate large quantities of APC and C-PC at a small fraction of the usual cost. In addition, we show that these fluorochromes can be readily coupled to monoclonal antibodies and used in three-color flow cytometric analysis of activated Peyer's patch B lymphocytes.

Materials and methods

Monoclonal antibodies The following FITC, biotin or APC conjugates of monoclonal antibodies were prepared in this laboratory from saturated ammonium sulfate precipitations of serum-free hybridon~a supernatants: 10-4.22, MEL-14 attd RA3-6B2, which are directed against lgD, the lymph node-specific homing receptor, and B220, respectively (Oi and Herzenberg, 1979; Coffman, 1983; Gallatin et al., 1983). Rabbit anti-rat Ig was purified by extensive absorption of serum with mouse IgG-Sepharose in order to remove any anti-monse cross-reactivity, followed by elution from a rat IgG-Sepharose column. Phycoerythrin-streptavidin was obtained from Biomeda Corp., Foster City, CA.

Source and purification of allophycocyanin Spirulina platensis was obtained in a dry, powdered form (not tablets), from a health food store (New Pioneer COOP, Iowa City, IA; distributed by Frontier Herbs, Norway, IA). The method of isolation of APC and C-PC from Spirulina has been modified from that of Hardy (1986), and involves the lysis of the bacterial cell walls with lysozyme, followed by the separation of C-PC from APC by elutica from an hydroxylapatite column using increasing phosphate concentrations. 10 g (dry weight) of the Spirulina powder were suspended in 500 ml of 0.1 M phosphate buffer (pH 7.0) containing 100 lag/ml lysozyme (Sigma Chemicals, St. Louis, MO) and 10 mM EDTA. The suspension was incubated at 37°C overnight in a shaking water bath and then centrifuged for 40 rain at 1500 × g. The supernatant was precipitated with 50~ saturated ammonium sulfate at 4°C, redissolved in a minimal volume of 1 mM phosphate buffer (pH 7.0) con-

taining 0.1 M NaCI, and dialyzed ext.eusively against the 1 mM phosphate buffer. The sample was centrifuged in a 50 Ti rotor (Beckman, Palo Alto, CA) at 85000 × g for 1 h to remove any remaining fine green precipitate, resulting in a brilliant blue solution. In order to purify and separate APC from C-PC, the extract was applied to a !0 cm × 2.5 cm hydroxylapatite (Bio-Rad, Richmond, CA) column equilibrated with 1 mM phosphate buffer. Upon addition to the column, all of the colored material remained bound to the top o n e - f i ~ of the column. The column was then washed extensively with 1 mM phosphate bt~ffer. C-PC and APC were separated from each o~her using a 10-70 mM continuous phosphate gradient (containing of 0.1 M NaCI, 1 mM sodium azide). C-PC eluted at the lower phosphate concentrations, followed by APC; any APC that remained bound to the column was then eluted with 250 mM phosphate (also with 0.1 M NaCI, 1 mM sodium azide). The material eluted from the column was monitored visually and with an ISCO model UA5 absorbance/fluorescence detectoz with a model 1133 multiplexer-expander, reading simultaneous absorbance at 280 um and 660 nm. 3 ml fractions were collected. The appropriate fractions were pooled, concentrated using vacuum dialysis or Aquaeide (Calbiochem, La Jolla, CA), and dialyzed against 100 mM phosphate buffer (pH 6.8), supplemented with 50 mM NaCI (without azide). Protein concentrations in m g / m l were determined using the specific extinction coefficient of 6.35 for APC at 650 urn, pH 7.0 (Brown and Troxler, 1977) and 7.0 for C-PC at 620 nm, pH 7.0 (Hardy, 1986).

Coupling of phycobiliproteins to antibodies The co./~lht~ ~:~ ~tiluphycocyauhl to antibodies is similar to a method previously described (Hardy, 1986), and involves three separate reactions. The first couples the heterobifun':tional crosslinker succinimldyl 4-(p-maleimidophenyl) butyrate (SMPB) (Sigma) to lysine e-amines or primary a-amines of the antibody. The second reaction involves the thiolation of APC with 2-iminothiolane (2-IT) (Sigma) which introduces -Stl groups on APCs N-terminal amines. The thiolated APC is then reacted with the SMPB-coupled antibody,

and the subsequent cross-linking results in the APC-conjugated antibody. Since the optimal ratio of SMPB to antibody varies for different monoclonals, three different ratios are usually done when conjugating a monoclonal antibody for the first time. Once the optimal ratio is determined, testing of ratios is no longer necessary. The detailed conjugation method follows. The purified antibodies were adjusted to a concentration of 4--5 mg/ml, and dialyzed against 100 mM phosphate, supplemented with 50 mM NaC1, pH 6.8 (coupling buffer), in order to remove any azide. The azide-free antibody was then divided into three 0.5 mg fractions. SMPB was dissolved in dimethyl formamide at 12.5 mg/ml, and either 1/~1, 2 pl or 4/~1 ef the SMPB solution was added to each of the antibody fractions. The additions were made quickly, and the solutions were vcrtexed and allowed to incubate for 2 h on a rotator at room temperature. While the antibody-SMPB solution was reacting, APC was thiolated by treatment with 2iminothiolane (2-IT). The optimal ratio of thiolated APC to SMPB-coupled antibody was determined to be 0.8 mg of 2-IT-APC to 1 mg of SMPB-antibody. Therefore, since 1.5 mg of total antibody were to be coupled to APC, 1.2 mg total of APC at 4 m g / m l in coupling buffer were reacted with 140 pl of 2-1T (10 m g / m l in coupling buffer) for 1 h at 25°C. The antibody and PbP incubations were timed so that both the antibody reactions and APC reaction ended at the same time. The antibody preparations were then centrifuged for 5 rain in an Eppendorf 5414 centrifuge to remove any precipitate and dialyzed ag:~inst two changes of PBS (pH 6.0) to remove any unreacted SMPB. Likewise, the 2-1T-reacted APC was dialyzed against two changes of PBS (pH 7.2) to r,mov¢ any unreacted 2-IT. 0.4 mg of 2-1T-APC were then mixed with 0.5 mg of SMPB-antibody and placed on a rotator at room temperatu.'e. The reactions were checked by centrifugation for the appearance of any appreciable precipitate every hour. The reactions were quenched b y the addition of N-methyl-maleimide (final concentration of 10 mM) after 4 h or earfier if a noticable precipitate had formed. The reacted mixture was then centrifuged and dialyzed against two changes of 1 mM phosphate, 0.1 M NaCI, and 1 mM

12 azide, pH 7.0. The equilibrated mixture was then chromatographed on a small hydroxylapatite column (1 mi bed volume) in order to separate conjugated and unconjugated antibody. All of the colored material bound to the uppe2" 1/10 of the column and the column was washed extensively with 1 mM phosphate. Colored fractions were then eluted with 10 raM, 50 raM, 100 mM and 250 mM phospha',e; all of these phosphate buffers contain 0.1 M NaCI and 1 mM aside. All of the colored material that eluted at a given phosphate concentration was pooled. The antibody activity of the different pooled sets was determined by FACS analysis of appropriate antigen-positive cells.

Cell staining For single color staining, or three color staining with directly coupled reagents, 5 × 105 cells were incubated with 2 5 / t l of labelled antibody(ies) in V-bottom 96 well plates for 20 rain, resuspended in 100 p l of PBS supplemented with 2% bovine serum albumin (BSA), underlayered with three drops of 10% BSA, and centrifuged for 3 rain. The cells were then resuspended in 0.5 ml of PBS supplemented with 2% BSA. Just prior to analysis, 0.5/tg of propidium iodide (Sigma) was added for dead cell gating (Sasaki et al., 1987). For muiticolur stainings utifizlng unconjugated rat monoclonals, cells were first incubated in 25/tl of unconjugated rat monoclonal antibody for 20 rain. The cells were then diluted with BSA-supplemented PBS, centrifuged through a 10% BSA undeflayer, and stained with 25 pl of APC anti-rat lg. After 20 min, 5 pl of normal rat serum was added to block any residual anti-rat binding sites. After washing, the cells were incubated with 25 pl of FITC- and biotin-conjugated antibodies supplemented with 5,% ~ormal rat serum, incubated, and washed as before. Finally, the cells were incubated with 25 pl of PE-streptavidin, and then washed and resuspended in PBS as with single stage staining.

Deter,,;,~otion of absorption, excitation and emission spectra Absorption curves were obtained from a Beckman model 25 double beam scanning spectrophotometer. Fluorescence excitation and emission

curves were obtained with a Perkin-Blmer 650-10S fluorescence spectrophotometer. Excitation and emission curves were obtained with the reading slit width set at 2 nm.

Determination of quantum yield The quantum yields of C-PC and APC were determined according to the methods of Parker (1968). The concentrations of the C-PC and APC samples, as well as the creysl fast violet standard, were adjusted so that their absorption at the excitation wavelength of 610 nm was 0.100 + 0.001. The quantum yield of the creysl fast violet standard was 0.98:1:0.07 (Dale and Teale, 1970). Fluorescence emission curves were obtained on a SLM-Aminco SPF-500C spectrofluorimeter and inte~ated with the accompanying software.

Determination of relative molecular weight of APC preparations Monomeric APC ((a, fl)l was produced by dissociating native trimeric APC ((a, ~)3) according to the method of MacColl et al. (1980) by extensive dialysis against 0.1 M sodium acetate buffer, pH 3.9. Sucrose density gradients (from 5% to 30%) were prepared in 14 × 95 mm nitrocellulose ultracentrifuge tubes with 0.1 M phosphate, 0.1 M NaCI, p H 7.0 for the purified APC, or 0.1 M sodium acetate, 0.1 M NaCI, p H 3.9 for the monomeric APC. 200 pl of a 4 m g / m l solution of each PbP sample was overlaid on the respective gradient, and centrifuged in a SW 40 Ti rotor (Beckman) at 160000 × g for 22 h at 4°C.

Flow cytometry A dual laser five parameter FACS 440 flow cytometer (Becton Dickinson) was used in these experiments. FITC and PE were excited by an Innova 90-5 argon ion laser emitting at 488 nm and APC and C-PC were excited with a dye laser at 595 nm using rhodamine 6G pumped by an Innova 90-5 argon laser (both from Coherent Lasers, Pe.lo Alto, CA). Emission from APC and C-PC was collected through a 660/20 band pass filter using a red sensitive, R1477-04 Hamamatsu photomultiplier tube (Becton Dickinson). FITC, PE and propidium iodide emissions were passed through 530/30, 575/26 and 625/35 band pass filters, respectively. Data from 10000 cells were

collected using logarithmic amplifiers (four decades for 256 channels) in list mode on a VAX 11/750 and analyzed with the F A C S / D E S K program (kindly supplied by Wayne Moore, Stanford University).

Results Lysis and saturated ammonium sulfate precipitation of 10 g of Spirulina results in a precipitate which contains A P C and C-PC. This mixture is redissolved and applied to an hydroxylapatite column. A P C and C-PC are separated from each other by eluting with increasing phosphate concentrations. The binding and elution of C-PC and

APC is followed visually as well as by monitoriv~:~ absorbance at 660 nm and 280 Contain/hating proteins, detected by absorbance at 280 nm, were washed from the column with two bed volumes of 1 m M phosphate (data not shown). After the phosphate gradient was begun, the purp,~e colored C-PC eluted at relatively low concentrations of phosphate and the blue colored A P C at .higher phosphate concentrations (Fig. 1). Complete elution of A P C required stepping the phosphate concentration to 250 raM. In order to determine t~e identity of the eluted material, absorbance spectrum curves obtained from selected fractions were compared to the published spectra of pure C-PC and A P C (Kufer and Scheer, 1979; Oi et al., 1982). Fraction number 50, which corresponded to

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Fig. 1. Elution profde of C-PC and APC. Protein was eluted from an hydroxylapatite column with a 10-70 mM continuous phosphate gradient, followed by 250 mM phosphate. Phycobiliprotein concentration was monitored by reading absorption at 660 run. Effectiveness of separation was assessed by comparing the absorbance spectra of selected fractions with published spectra. Fraction

50 has a peak absorbance at 620 nm, which corresponds to pure C-PC. Fraction 108 has two absorbance peaks, one at 620 nm (C-FC) and one at 650 nm (A.'~). Fraction 117 has an absorbance curve which corresponds to that of pure APC. Fractions 45 through 65 (for C-PC) and fractions 116 through 135 (for APC) were pooled. Monitoring of A280demonstrated that contaminatin8 proteins were removed from the column with two bed volumes of the 1 mM phosphate. In addition, the elution profde measured by A280closely paralleled that of the profde at 660 nm (data not shown).

14

the first peak eluted, was essentially pure C-PC, having a n a b s o r b a n c e peak at 620 nm. Significant c o n t a m i n a t i o n of A P C b y C-PC was present in the leading edge of the second peak, as shown b y the shape of ~ e spectral curve of fraction 108. However, b y fraction 117, which corresponded to the apex of the second peak, a n absorbance spectrum characteristic of pure A P C was obtained. T h e fractions which contained the first peak (C-PC) were pooled as well as all of the colored fractions after fraction 116 (APC). Both pooled fractions were concentrated a n d dialyzed against 100 m M p h o s p h a t e with 0.5 M NaCI. F r o m the original 10 g of powdered Spirulina, approximately 80 m g of p u r e C-PC a n d 40 m g of pure A P C were isolated, at a cost o f approximately $0.03/mg. Additional A P C can b e o b t a i n e d b y rechromatographing the fractions corresponding t o the leading portion of the second (APC) peak o n a regenerated hydroxylapatite column (dat:~ c o : shown_). Fluorescence excitation a n d emission spectra of the purified C-PC a n d A P C were o b t a i n e d a n d were in excellent agreement with previously p u b fished excitation a n d emission curves for C-PC a n d A P C (Fig. 2) (Bonsslba a n d Richmond, 1980; MacColl et al., 1980). T h e b r o a d excitation curves

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700 500 600 700 Wevete,gr,(nn0 Fig. 2. Fluorescence.excitation and emission spectra of purified C-PC and APC. The fluorescence spectral properties of the pooled fractions of PC and APC were determined. Emission curves are shaded gray. A : PC has excitation maxima at 590 nm and 620 run, and an emission maximum at 650 nm. B:APC has excitation maxima at 650 nm and 610 rim, and an emLo.~ionmaximum at 660 rim. These fluorescence p~'ofilesare identical to those previously shown for APC and C-PC.

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Fig. 3. Sucrose density g, adient centrifugafion analysis of purified trimeric (a,/~)3 APC and dissociated monomeric (a, ~) APC. Ultracentrifugation of an acid treated, monomeric APC standard resulted in two bands (A and B); centrifugation of purified APC resulted only in band C. Absorbance spectral curves of the isolated bands showed that band A was monomeric APC. Band B corresponded to a small amount of trimerle, native APC remaining in the monomeric APC preparation. The single band (C) of the purified, pooled APC had an absorbance spectrum identical with trimeric APC. Thus, the process of purification of APC from Spirulina yielded only trimeric APC, without any subunit dissociation.

o f each protein p e r m i t strong excitation b y a tunable dye laser or by the 633 n m fine of a helium-neon laser. I n addition, b o t h proteins have en~ssion peaks easily separable from the exciting wavelengths. T h e two phyeobiliproteins were further characterized b y determining their fluorescence q u a n t u m yield. T h e emission spectra of the proteins were c o m p a r e d to the emission spectra of a stand a r d solution of creysl fast violet. T h e absolute q u a n t u m yield of C-PC a n d two different batches of A P C were determined. O u r experimentally

determined quantum yield for C-PC (0.47 + 0.03) is in good agreement with previously published values of 0.51 and 0.52 for the quantum yield of C-PC (Dale and Teale, 1970; Grabowski and Gantt, 1978). In addition, the quantum yield determinations of two different batches of APC (0.62 4-0.04 and 0.69 + 0.05) agree well with the quantum yield measurements for APC of 0.68 detem~ned by Grabowski and Gantt (1978). Thus, the fluorometric properties of these isolated proteins are virtually identical to previously characterized APC and C-PC. The excitation and emission l~roperties of these ~roteins are determined not onl~ by the number and types of prosthetic groups attached to the protein core, but also by the state of association of the ¢-/~ subunits~ For example, the dissociation of trimeri~ APC ((a,/~)3) into monomeric APC (a, ~ ) causes the absorption maximum to shift from 650 nm to 615 nm and the emission maximum to shift from 660 n m to 641 nm (MacColl et al., 1980; Huang et al., 1987). Such dissociated material is not suitable for mult~color FACS analysis. Therefore, in order to determine if any of the purified material became ..'~ssociated during purification, sucrose density centfifugation was performed on the pur~Yied APC and a monomeric APC standard. The monomeric APC preparation, prepared by dialysis against acid, showed a major band and a smaller amount of relatively higher

molecular weight material (Fig. 3, band A and B). The purified APC showed no band other than that corresponding to the higher molecular weight material (band C). The identity, of the bands was then det~mined spectroscopically. Absorbance curves obtained on material from the isolated lower molecular weight band was confirmed to be monomeric APC, having an absorbance maximum at 615 nm (Fig. 3A); th~ higher molecular weight material (bands B and C) corresponded to trimeric APC, with its absorbance peak at 650 nm (Figs. 3B and 3C). No band corresponding to monomeric APC was detected in the sucrose density analysis of the original purifie~ twaterial. Thus, the APC purified in these experiments retained its trimeric subunit structure. The long term stability of APC and APC conjugates was ~ assessed. APC retains its trimeric form even after prolonged storage of various forms. The purified material has been stored as a concentrated (4 mg/ml) solution at 4 ° C or frozen at - 70" C, or as a saturated ammonium sulfate precipitate for over 1.5 years, with no appreciable subunit dissociation or change in absorbance spectrum curves. The most convertient form of storage is as a liquid so!ution (4 mg/ml) at 4 ° C that has been dialyzed against coupling buffer (without azide) and falter-sterilized. This APC form is immediately usablc for antibody coupling without additional dialysis. As with the purified material

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Fig. 4. Histogramsof normalmurinespleencellsstainedwith differentconjugatesof the pan B cell marker, anti-B220.A: directly APC-conjugatedanti.B220.B: unconjugatedanti-B220,followedby APC-conjugatedrabbitanti.ratlg. C: directlyFITC-conjugated anti-B220.Note that the stainingintensityof the directlyAPC-conjugateda~fibodyis comparableto that of the FITC-conjugated antibody.

the APC-antibody conjugates are also very stable. APC-coupled reagents prepared over 2 years ago using these methods still retain their trimeric structure and have had no loss of antibody activity (data not shown). The relative fluorescence intensity of the APCconjugated reagents have been compared to FITC-conjugated antibodies. Representative FACS histograms of murine spleen cells stained with the pan B cell marker RA3-6B2 (anti-B220), directly coupled to APC and FITC and the unconjugated monoclonal stained with an APC-coupled anti-rat If,, are shown in Fig. 4. Clear separation was achieved with the APC-coupled reagent, comparable to that obtained with the corresponding FITC-anti-B220 conjugate. Staining with the unconjugated monoclonal antibody and a second stage APC-anti-rat Ig is brighter than the directly coupled reagent, as would be expected with the signal amplification achieved with two-stage immunoflunrescence. One important advantage for APC-coupled reagents is their utility in both three- and fourcolor immunofluorescence analysis where the other fluorochromes are FITC, PE and Texas Red. The suitability of our APC-conjugated reagents was therefore tested in multicolor FACS analysis. Murine Peyer's patch cells were stained with FITC-anti-B220, biotinylated anti-lgD, and APCMEL-14 (which is directed against the lymph node-specific homing receptor gp~'0MeL'14). AS

shown in Fig. 5, these reagents define several distinct cell populations. Of particular interest is the relationship between IgD expression and homing receptor expression. Three-color analysis demonstrates that the IgD-negative B cells (gate I, on the contour plot) are homing receptor-negative (Fig. 5, histogram I). In contrast, the IgD-positive B cells (gate If) express high levels of homing receptors (histogram II). These results are consistent with previous work that has demonstrated an inverse relationship between activated cells and homing receptor expression (Reichert et al., 1983; Tung and Dailey, 1987). Thus, the APC-coupled reagents produced from p~wdered Spirulina are well suited for multicolor flow cytometric analysis.

Discussion

Phycobiliproteins provide useful reagents for multicolor FACS analysis. The emission peaks from different fluorochromes are readily distinguished from each other permitting the simultaneous detection of multiple cell surface antigens. Besides being easily coupled to m0nodonal and polyclonal antibodies, APC and C-PC have the additional advantage of being easily purified from cyanobacteria. The main reasons hindering the widespread use of allophy¢ocyanin or phycocyanL~ as fluorescent probes have been both the lack of an available source of blue-green algae and the

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Fig. 5. ~ o r analysisof activatedB cells in murine Peyer'spatches. Cellswerestained with FITC-anti-B220,PE-anti-lgDand APC-MEL-14(a monoclonalthat recognizesthe lymph node-specifichomingreceptor). 18D-negativeB cells (gate 1) are homing receptor-negative(histogramI). IgD-positiveB ceils(gate 11)are predominantlyhomingreceptor-positive.

high cost of purified material. Health food stores are a convenient source of low cost Spiruiina platensis from which these phycobiliproteins can be easily isolated. C-PC and APC are separated from bacterial cell lysates by elution from an hydroxylapatite column using an increasing phosphate gradient. The purity of the fractions was assessed by comparing the absorbance spectra with those previously pubfished for these proteins. Although we recommend checking the relative purity of the final pooled preparation, good separation can be obtained by pooling all of the first purple colored peak (C-PC) and all of the blue material after the apex of the second peak (APC). Alternatively, a greater yield of APC can be obtained by passing the entire pooled APC peak through the regenerated hydroxylapatite column a second time. Dissociation of the isolated APC from its native trimeric form ((a,/3)3 ) to its monomeric form (a,/3) profoundly changes its excitation and emission curves, as well as decreases the number of chromophores capable of being coupled to antibodies. Dissociation results in proteins that are unsuitable for FACS analysis. Therefore, the state of polymerization of our purified material was assessed. Sucrose density gradient centrifugation of the purified APC product demonstrated a single blue band, which was shown to be the desired trimeric APC (Fig. 3). Similar analysis showed that the purification of C-PC did not cause its dissociation into monomers (data not shown). The fluorescence excitation and emission curves of these two phycobiliproteins also corresponded exactly to that of trimeric APC and C-PC (Fig. 2). These spe¢tr~ curves demonstrate the suitability of these proteins for ;~e in immunofluorescence and flow cytometry, with good excitation being achieved with either a standard dye laser or a helium-neon laser. APC is especially useful, since its emission (at 660 rim) can be adequately separated from that of Texas Red (using 660/20 and 630/22 band pass filters, respectively), such that they can be used in four-color FACS analysis. PC-coupled reagents have also been prepared and exhibit good immunofluorescence staining (data not shown). Although PC-coupled reagents are useful in three-color FACS analysis when used with FITC and PE, the emission maximum of PC (650 nm) precludes its simultaneous use with either

Texas Red or APC. It should be noted that not all commercial Spirulina platensis preparations can be used for APC and PC purification. Algae sold in tablet form were found to yield completely dissociated monomeric proteins, presumably due to denaturing that occurred during processing. Thus, when evaluating a new source of Spirulina, it is important to check at least the absorbance spectra of the final purified phycobiliproteins in order to ensure that the spectral profde corresponds to that of trimeric ((a,/~)3) proteins. The conjugation of APC and C-PC involved the reaction of antibodies with the heterobifunetional linker SMPB and separately reacting APC or C-PC with 2-1T. These two reacted species are then mixed and allowed to combine to form the stable conjugated reagent. Similar conjugates have also been prepared using the heterobifunctional linker succlnimldyl 4-(?7-maleimidomethyl) cyclohexane-l-carboxylate (SMCC) (Pierce, Rockford, IL) with equally good results. It is of parfica.lar importance to maintain a high concentration of antibody and phycobih'protein throughout these conjugation procedures. Excessive dilution of either reactant results in conjugates that stain poorly and have low titers. Optimally coupled reagents obtained with this conjugation method stain cells with a high intensity, provide clear separation between positive and negative cell populations, and are ~table for at least 2 years. The APC-coupled anti-rat Ig second stage reagent has an add;.tional advantage in that it provides a relatively simple means of doing three color staining in which one of the reagents is an unconjugated rat monoclonal antibody (Fig. 4C). In summary, Spirulina platensis, in bulk powdered form, is a convenient and inexpensive source of cyanobacteria from which can be purified allophycocyanin and C-phycocyanin. These phycobillproteins are easily isolated without any subunit dissociation or change in spectral properties. Both proteins have been successfully coupled to numerous monoclonal and polyclonal antibo~Iies. These fluorescent antibody reagents are stable and consistently provide bright and specific immunofluore~scence staining. The widespread use of allophycocyanin should facilitate multiparameter flow cytometry and cell sorting for more precise and in-depth studies of cell surface antigens.

Acknowledgements T h i s w o r k was s u p p o r t e d b y N I H G r a n t A I 22730 a n d A C S G r a n t 1N-122F. M o r r i s Dailey is a fellow o f t h e J o l m A. H a r t f o r d F o u n d a t i ~ t . T h e a u t h o r s t h a n k Dr. D a ~ d Parks ( S t a n f o r d U n i v e r s i t y ) for h e l p f u l dis~cussions r e g a r d i n g p h y c o b i l i p r o t e i n purification.

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